Intrauterine stress can induce long-lived changes in function that predispose the fetus for disease throughout life. Maternal hypoxemia due to high altitude living, placental insufficiency, and smoking puts infants of certain populations at risk o developing pulmonary hypertension, high altitude pulmonary edema, and increases the risk for development of idiopathic pulmonary hypertension later in life. Gestation at high altitude causes a variety of pulmonary vascular malformations in fetal sheep including suppressed endothelium dependent relaxation, altered myocyte growth, and reactivity that place them at risk of developing hypertension after birth. Indeed, high altitude gestation and birth exaggerates hypoxia induced pulmonary vasoconstriction in 2 week old sheep and results in significant penetration of pulmonary hypertension. Sheep are therefore a relevant model for understanding novel pathways and mechanisms associated with fetal origins of pulmonary vascular disease because hypoxemia due to high altitude gestation recapitulates disease found in humans. The fundamental focus is that ryanodine receptors (RyR) are central to the process of pulmonary arterial constriction in response to acute alveolar hypoxia, which can be dysregulated in individuals with pulmonary hypertension. Three RyR isoforms are known, and all three are vital to the intrinsic vasoconstrictor response to acute hypoxia in pulmonary arteries. RyR channelopathies actively participate in the pathogenesis of neuronal, skeletal muscle, cardiac, and vascular diseases and yet their role in fetal programming of pulmonary vascular disease is unknown. The central hypothesis of this project is that high altitude gestation will suppress RyR-mediated local Ca2+ sparks and global Ca2+ responses due to acute hypoxia and that these responses will be manifested by reductions in RyR expression. We propose this counterbalances mechanisms that would increase vascular contraction to acute hypoxia. This would help reduce the extent of pulmonary hypertension due to long-term intrauterine hypoxia. The central hypothesis is based on published and preliminary studies performed with full-term fetal sheep. We plan to test our central hypothesis by pursuing two Specific Aims. Specific Aim 1 will determine whether high altitude gestation suppresses the expression of the three RyR isoforms. Specific Aim 2 will determine the corresponding reduction in RyR mediated Ca2+ responses and pulmonary vasoconstriction in response to acute hypoxia. The hypotheses will be examined by performing molecular, histochemical, and functional studies in pulmonary arteries from term-fetal lambs. With regards to the expected outcomes, the work proposed is expected to identify the influence of long-term intrauterine hypoxia on RyR function in the fetus. These studies are expected to fundamentally advance the field of pulmonary vascular biology by associating RyRs with pulmonary vascular disease. Such results will provide an important positive impact, because RyRs are highly likely to provide novel targets for therapeutic interventions in the treatment of pulmonary vascular disease. PUBLIC HEALTH RELEVANCE: The investigations we propose help provide a mechanistic basis for the role of long-term intrauterine hypoxia in the development of pulmonary hypertension in the newborn. The goal of these studies is to associate ryanodine receptors with disease pathogenesis. Such results will provide an important positive impact, because ryanodine receptors are highly likely to provide novel targets for therapeutic interventions.